Eukaryotic cytosine methylation has been involved in such diverse biological functions as gene repression, Xchromosome inactivation, genome imprinting, and replication timing. DNA cytosine methylation is essential for the normal development of plant and mouse. Moreover, alterations in genome methylation patterns contribute to the genesis of human cancers. Our long-term goal is to understand the two sides of mammalian DNA methylation from a structural standpoint: a class of enzymes called DNA cytosine-5-rnethyltransferases (DNMTs) that methylate DNA and a class of proteins containing a methyl-CpG-binding domain (MBD) that bind to methylated DNA. In this application, his immediate focus is on three mammalian enzymes (Dnmtl, Dnmt2, and Dnmt3b), and five methyl-CpG binding proteins (MeCP2, MBD1 to MBD4). Two human genetic diseases (ICF and Rett syndromes) have been attributed directly to mutations in Dnmt3b and MeCP2. Our specific aims are: 1. to determine the structure of full-length human Dnmt2; 2. to determine several domain structures of mouse Dnmt3b; 3. to determine several domain structures of human Dnmt1, and 4. to determine the structures from one of five MBD domain-containing proteins and its complex with the methylated CpG-containing DNA.